The general aim of this project is the elucidation of the mechanisms by which genes are transcribed in eucaryotic cells. The main emphasis is reconstitution of transcription in vitro, in particular the study of the structure and function of each of the polypeptide components of eucaryotic RNA polymerases. The experimental method proposed here involved the use of specific antibodies. Specific functions of the enzymes and their subunits may be altered by antibody binding and blocking specific sites in the macromolecule. We plan to generate a collection of these probes by the monoclonal antibody technique. The effects of subunit-specific antibodies on the activity and transcriptional specificity of enzymes I, II and III will be tested by a variety of methods in order to probe the role of the affectee subunit. In addition we propse to use the antibodies as tools in the isolation of RNA polymerase mutants, studies on enzyme structure, enzyme synthesis and preliminary studies towards the isolation of the RNA polymerase genes.